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目的:探讨高糖和不同浓度胰岛素对大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖的影响及可能机制。方法:组织贴块法原代培养大鼠胸主动脉VSMCs,用高糖和不同浓度胰岛素诱导细胞增殖。实验设对照组、高糖组、高糖和不同浓度胰岛素组。采用四甲基偶氮唑盐[3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide,MTT]比色法测定VSMCs增殖;免疫细胞化学技术检测VSMCs增殖细胞核抗原(proliferatingcell nuclear antigen,PCNA)含量;RT-PCR技术检测VSMCs中PCNA mRNA的表达量;NO试剂盒检测培养基上清液中NO含量。结果:高糖能诱导VSMCs的增殖,而胰岛素则呈剂量依赖性地协同高糖诱导VSMCs增殖,最大作用效果出现在300 mU/L胰岛素培养的第5天;高糖可以使PCNA蛋白含量增加,而高糖和高浓度胰岛素(300 mU/L)合用则可使PCNA蛋白含量显著增加;高糖可使PCNA mRNA表达量增高,而高糖和高胰岛素(300 mU/L)合用则可使PCNA mRNA表达量显著增高;高糖可使NO含量降低,而高糖和高胰岛素(300 mU/L)合用则可使NO含量显著降低。结论:胰岛素呈剂量依赖性地协同高糖促进VSMCs增殖,使VSMCs PCNA蛋白含量和PCNA mRNA表达量增高,并协同高糖降低NO的产生。
Objective: To investigate the effects of high glucose and different concentrations of insulin on the proliferation of rat vascular smooth muscle cells (VSMCs) and its possible mechanism. Methods: Rat primary VSMCs were primarily cultured by tissue patch method. High glucose and different concentrations of insulin were used to induce cell proliferation. Experimental set control group, high glucose group, high glucose and different concentrations of insulin group. The proliferation of VSMCs was determined by MTT assay and the proliferation of VSMCs was detected by immunocytochemistry (proliferating cell nuclear antigen, PCNA). The expression of PCNA mRNA in VSMCs was detected by RT-PCR. NO was detected by NO kit in culture supernatant. Results: High glucose could induce the proliferation of VSMCs, while insulin induced VSMCs proliferation in a dose-dependent manner. The maximum effect was found on the 5th day of 300 mU / L insulin culture. High glucose could increase the content of PCNA protein, However, the combination of high glucose and high concentration of insulin (300 mU / L) could significantly increase the content of PCNA protein; high glucose could increase the expression of PCNA mRNA, while high glucose and high insulin (300 mU / L) The expression of NO was significantly increased. High glucose led to the decrease of NO content. However, the combination of high glucose and high insulin (300 mU / L) could significantly reduce NO content. CONCLUSION: Insulin promotes the proliferation of VSMCs in a dose-dependent manner with high glucose, and increases the expression of PCNA protein and PCNA mRNA in VSMCs. In addition, high glucose can reduce the production of NO.